Databases

The following databases were used to retrieve data.

Table 15: List of databases

MGI_3.43-mouse genome informatics http://www.informatics.jax.org/

National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/

Ensembl http://www.ensembl.org/index.html BasicLocalAlignmentSearchTool_BLAST

(NCBI) http://blast.ncbi.nlm.nih.gov/Blast.cg

- 30 - 5. Methods

5.1. Molecular biology methods

5.1.1. Nucleic acid isolation

5.1.1.1. Small-scale plasmid purification

For small-scale plasmid purification the buffer system of the PureLink^®HiPure Plasmid Midiprep or PureLink^®HiPure Plasmid Filter Midiprep kits was used. 1.5 ml of a bacterial overnight (O/N) culture was transferred to a 1.5 ml reaction tube and the cells were pelleted by centrifugation for 30’’ at 13,000 rpm. The supernatant was discarded, the pellet was resuspended in 200 µl buffer R3 supplemented with RNase (see chapter 5.1.1.3) and vortexed in short pulses until it was visibly homogeneous. After the addition of 200 µl buffer L7 the sample was incubated for 5’ at room temperature (RT). Hereafter, 200 µl buffer N3 were added and the sample was inverted until the solution was homogeneous. The cell debris was pelleted by centrifugation at 13,000 rpm for 10’ and 500 µl of the supernatant was transferred to a new 1.5 ml reaction tube containing 1 ml 99 % EtOH. The DNA was precipitated at -20°C for 1 h and pelleted by centrifugation for 30’ at 13,000 rpm at 4°C. The pellet was washed with 70 % ethanol (EtOH), centrifuged for 10’ at 13,000 rpm and dried at 55°C for 10’ and finally resuspended in sterile-filtered DNase-free ddH2O in an appropriate volume (50-100 µl) for 10’ at 42°C and 1,400 rpm on a heating block.

5.1.1.2. Medium-Scale Plasmid Purification

Medium-scale plasmid purifications were performed using the PureLink^®HiPure Plasmid Midiprep or PureLink^®HiPure Plasmid Filter Midiprep kit according to the manufacturer’s instructions. The plasmid DNA was solved in sterile-filtered DNase-free ddH2O. To increase plasmid purity an additional precipitation step was performed. Hence, the plasmid DNA was diluted to a final volume of 500 µl and either 1 volume-unit isopropyl-alcohol or 2.5 volume units 99 % EtOH were added. After thorough mixing by inverting, the DNA was precipitated O/N at -20°C and pelleted by centrifugation at 13,000 rpm for 30’. The pellet was washed, dried and resuspended as described in chapter 5.1.1.1. Plasmid stocks were stored at -20°C.

Methods

- 31 - 5.1.1.3. Isolation of genomic DNA

For isolation of genomic DNA (gDNA) mouse tail biopsies (see chapter 5.5.2) were incubated overnight (O/N) in 400 µl STE-buffer supplemented with 25 µl of proteinase K (10 mg/ml). Thereafter, the samples were briefly vortexed and centrifuged for 10’ at 13,000 rpm to remove tissue debris. 400 µl of the supernatant were transferred into a fresh reaction tube containing 1 ml cold 99 % EtOH. The samples were briefly vortexed and the DNA was precipitated for 10’ at -20°C, pelleted for 25’ at 13,000 rpm, washed with 500 µl 70 % EtOH and centrifuged again for 10’ at 13,000 rpm. Finally, the DNA was dried for 10’

at 55°C and resuspended in 125 µl ddH2O on a heating block for 10’ at 42°C and 1,400 rpm.

5.1.1.4. Isolation of total RNA from cell culture

Total RNA from cells was isolated using TRIzol-reagent according to the manufacturer’s instructions. In short, the cells were washed with cold sterile 1 x PBS and 1 ml TRIzol was added to the cells. To ensure the integrity of RNA the following steps were performed on ice unless stated otherwise. The samples were first transferred into 2 ml reaction tubes and vortexed for 2’ on the highest setting. Afterwards the samples were kept at RT for 5’. After the addition of 200 µl chloroform the samples were vortexed for 15’’ and incubated for another 3’ at RT. The solution was centrifuged for 10’ at 13,000-14,000 rpm and 4°C. The upper phase was transferred to a fresh 1.5 ml reaction tube containing 1 ml 99 % DNAse/RNAse-free EtOH and inverted several times to ensure proper mixing. Subsequently, the RNA was precipitated O/N at -20°C, pelleted by centrifugation at 13,000 rpm for 30’ and 4°C and washed two times with 500 µl 70 % DNase/RNase-free EtOH. Finally, the pellet was dried for 10’ at 55°C, resuspended in 20 µl DNase/RNase-free H2O and solved for 10’ at 56°C on a heating block. Until further use the RNA was stored at -80°C.

5.1.1.5. Isolation of total RNA from murine skin samples

The isolation of RNA from mouse skin, skin biopsies and nude mice allografts (see chapters 5.2.11, 5.5.7 and 5.5.8) was performed using the RNeasy Fibrous Tissue kit according to the manufacturer’s instructions, with some modifications. In brief, a maximum of 30 mg tissue was minced using sterile scalpels and transferred into a 1.5 ml reaction tube containing 300 µl buffer RLT supplemented with 3 µl of the supplied β-mercapto-EtOH. The tissue was homogenized on ice using a tissue homogenizer for 90’’. After each sample the

- 32 -

tissue homogenizer was cleaned and rinsed in sterile, RNase-free ddH2O two times and once with RLT-buffer. The homogenate was processed as stated in the manufacturer’s instructions.

After the elution of the RNA a second DNase-treatment was included. For this purpose, the eluate was supplemented with 3.22 µl RDD-buffer and 1.4 µl DNase I and subsequently incubated for 1 h at 37°C. To inactivate the DNase the solution was incubated for 5’ at 65°C.

Afterwards 500 µl 99 % DNase/RNase-free EtOH was added to the solution and the RNA was precipitated at -20°C O/N. Finally, the RNA was pelleted and solved (see chapter 5.1.1.4). Until further use the RNA was stored at -80°C.

5.1.1.6. Reverse transcription of RNA (cDNA-synthesis)

For cDNA synthesis 2 µg RNA in a final reaction volume of 20 µl were reversely transcribed using the SuperScript II Reverse Transcriptase System. 250 ng hexamers were incubated with the RNA for 10’ at 70°C. Afterwards 1 x 1^st Strand Buffer, 10 mM dithiothreitol (DTT) and 0.5 mM deoxynucleotides (dNTPs) were added and the mixture was incubated for 10’ at RT. After pre-warming to 42°C for 2’, 1 µl of SuperScript II (200 U/µl) was added and the mixture was incubated for 1 h at 42°C. Finally, the reaction was stopped at 70°C for 10’. Assuming the reverse transcription reaction is 50 % efficient the final concentration of cDNA was 50 ng/µl.

5.1.1.7. Photometric quantification of nucleic acids

To quantify the concentration of DNA or RNA, 1 µl of the sample solution was diluted with 200 µl DNase/RNase-free ddH2O. The concentration was measured on a photometer by determination of the optical density (OD) at A260. The final concentration was calculated according to the formula:

= 260 × 50 ( ) 40 ( )

with c being the concentration (ng/µl). Additionally, the OD at A280 was measured to determine the amount of protein. With these values the purity was calculated as the ratio of OD260 and OD280. A ratio between 1.8 and 2.0 was considered sufficiently pure.

Methods

- 33 - 5.1.2. Polymerase-Chain-Reaction (PCR)

5.1.2.1. PCR-based genotyping of mouse tail gDNA and semiquantification of cDNA For PCR-based amplification of gDNA or cDNA the following reactions were carried out in a 10 or 20 µl reaction volume. For the primer combinations mPTCNx_f/mPTCNx_R/mPTCwt_R.2, EIIa-Cre-F/EIIa-Cre-R and Exon7-F/Neo-R the following conditions were applied:

0.1 U Taq polymerase (Moltaq)

Amplification of Gapdh transcripts was conducted using the following settings, but the same reaction mixture:

The resulting amplification products were separated and visualized by agarose gel electrophoresis.

5.1.2.2. Quantitative Real-Time PCR (qRT-PCR)

Differential gene expression was analyzed by SYBR-green based quantitative real-time PCR (qRT-PCR). Primer combinations are listed in table 10. The following reagents were used in a total reaction volume of 10 µl.

2.5 ng template cDNA 1 x SYBR-green 0.4 µM forward primer 0.4 µM reverse primer

Gene expression values were calculated using the standard curve method. For this purpose, 5-fold serial dilutions from tissue or cells known to express the target gene, starting with 20 ng cDNA, were prepared and amplified by qRT-PCR. The experimental Ct values

- 34 -

were interpolated from the standard curve by nonlinear-regression using the semilog-line model of the GraphPad Prism 6. Afterwards, the expression values of each sample were normalized to the respective expression of the housekeeper genes 18S rRNA or TBP. For housekeeper quantification 0.8 pg or 0.25 ng cDNA, respectively, were used as template in the qRT-PCR reaction. The first dilution standard-series of the standard-curve contained 20 ng cDNA obtained from a tissue that was known to express the gene of interest. The first dilution for 18S rRNA expression measurement contained 80 pg cDNA. The samples were measured in triplicates and the data was analyzed using SDS 2.2 (and higher) and GraphPad Prism 6.

5.1.2.3. Agarose gel electrophoresis

To separate DNA by its size, agarose gel electrophoreses were performed. For this, agarose gels containing 0.5 to 2 % (w/v) agarose in 1 x TBE buffer were prepared by boiling for 2-3 min at 1,000 W in a microwave. After cooling down, the still liquid gels were supplemented with 5-7 drops of a 0.07 % ethidiumbromide solution. The electrophoresis chamber was filled with 1 x TBE buffer. To ease the gel-loading, the samples were either diluted with 10 x cresol solution or 6 x loading dye to obtain a final concentration of 1 x. In parallel to the samples an appropriate DNA ladder was loaded. The gels were run at a constant voltage of 80 to 150 V. For documentation a UV transilluminator was used.

5.1.3. Cloning techniques

5.1.3.1. Transformation of E.coli with plasmid DNA

Competent E. coli DH5α were thawed on ice and 50-100 µl of the cells were mixed with 50-100 ng of plasmid DNA. The remaining cells were immediately frozen and stored at -80°C. After two freeze-thaw-cycles the bacteria were discarded. The plasmid/bacteria mixture was incubated on ice for 20’ and subsequently subjected to a heat shock at 42°C for 45’’. Afterwards, the cells were incubated again on ice for 2’. After the addition of 500 µl super optimal broth with catabolite repression (SOC) medium the cells were incubated for 1 h at 37°C and 900 rpm. Finally, an appropriate volume of the cell suspension (usually 20 to 200 µl) was plated onto lysogeny broth (LB) agar plates containing the adequate antibiotic as a selection marker. The plates were incubated in an incubator O/N at 37°C. The next day single colonies were chosen for further applications.

Methods

- 35 - 5.1.3.2. Restriction hydrolysis

Restriction endonucleases cut DNA on specific sites according to their sequence. The recognition site usually consists of 4-8 palindromic nucleotides. In this work test-restriction hydrolysis and preparative restriction hydrolysis were performed. For the test restriction the following mixture was prepared, in which the buffer was chosen according to the manufacturer’s instructions:

100-250 ng plasmid DNA 0.1 U restriction enzyme

1 x buffer

ad 10 µl ddH2O

If necessary and possible, a double restriction was performed. If not, a sequential restriction was performed by increasing the total volume to adjust for different salt concentrations of the different buffer systems of up to 50 µl. If the buffers were not compatible the DNA was precipitated after the first restriction reaction and, once solved again, subjected to the second restriction reaction. The restriction reaction was usually carried out for one hour at the optimal temperature for each enzyme. If possible, enzymes were heat-inactivated afterwards. Finally, the complete sample was separated by agarose gel electrophoresis.

In case of preparative restrictions the amount of DNA was increased as needed and the amount of enzymes used was increased accordingly, following the manufacturer’s instructions. Also, the restriction reaction duration was increased to ensure maximal restriction to a maximum time of 16 h. All used enzymes were purchased from NEB (Ipswich, USA) or Invitrogen (Karlsruhe).

5.1.3.3. Isolation of DNA fragments from agarose Gels

DNA-fragments were excised from agarose gels with a sterile scalpel under 70 % UV-light to reduce the risk of UV-induced mutations (e.g. thymidin dimers). The DNA was cleaned-up using the QIAEX II Gel extraction kit according to the manufacturer’s instructions including all optional washing steps.

- 36 - 5.1.3.4. Ligation

During a ligation reaction two pieces of DNA with compatible restriction ends are being connected. The ligation reactions were performed by employing the T4 DNA Ligase system. The molar ratio of the respective insert to vector was 3:1. The molar ratio was calculated according the following formula:

= ×

× ( : )

A reaction with a total amount of 100 ng DNA was set up according to the ligation protocol below.

X ng vector DNA Y ng insert DNA 4 U T4 DNA Ligase

1x T4 Ligase buffer

ad 10 µl ddH2O

The ligation reaction was conducted at 4°C for at least 48 h, stopped by heat-inactivation for 10’ at 65°C and transformed into E.coli (see 5.1.3.1).

5.1.3.5. DNA sequencing

The nucleotide sequence of plasmids was determined by the Sanger sequencing method using the BigDye^® reagent and an ABI3500XL sequencing device. For sequencing the following reagent mixture and conditions were used.

1x BigDye buffer 1‘ 95°C

1x BigDye 3.1 30‘‘ 95°C

30 cycles

20-200 ng plasmid DNA 2.5‘ 60°C

100 pmol sequencing primer 5‘ 60°C

ad 10 µl ddH2O ∞ 8°C

The primers used for sequencing are listed in table 11. The obtained electropherograms were evaluated and the obtained sequences were analyzed using the freely available software BioEdit.

5.1.3.6. Site-directed mutagenesis

The QuikChange II directed mutagenesis kit or the QuikChange II XL site-directed mutagenesis kit were used to induce point mutations in plasmid DNA as described in

Methods

- 37 -

the manufacturer’s instructions. The specific PCR reagent mixtures and conditions are described in the respective chapters. DpnI restriction was performed immediately after the respective PCR. For transformation of the E.coli strain XL-10 gold SOC-medium was used.

5.1.3.7. Generation of Smo expression plasmids

The plasmid pHAGE mSmo^W113Y mCherry carrying a point mutation in the Smo CRD (W113Y) was a kind gift from Dr. Adrian Salic, Harvard Medical School, USA (Nedelcu et al. 2013). To generate the pMSCV mSmo^W113Y mCherry plasmid (see Appendix) the mCherry-tagged mSmo-sequence from pHAGE mSmo^W113Y mCherry plasmid was amplified using the primer pair SmoKlon1AF/SmoKlon1BR and Phusion High-fidelity polymerase in a 20 µl reaction volume according to the following protocol:

50 ng template plasmidDNA Hotstart 98°C

0.5 µM SmoKlon1AF (forward Primer) 10‘‘ 98°C

35 cycles 0.5 µM SmoKlon1BR (reverse Primer) 30‘‘ 60°C

0.2 mM dNTP mix 2‘ 72°C

1x HF buffer 10‘ 72°C

2.5 mM MgCl2 ∞ 8°C

0.05 U Phusion High-fidelity polymerase

The amplified 3,635 bp fragment was purified by agarose gel extraction (see chapter 5.1.3.3) and eluted in 30 µl ddH2O. Next, the DNA was restricted by BclI at 50°C O/N and NotI at 37°C for 2 h. The DNA was separated on a 0.7 % agarose gel and the 3,559 bp fragment was excised, purified and subcloned into the pMSCVpuro vector after BglII/NotI digestion (kindly provided by Dr. Michael Engelke). The insertion of the respective fragments was verified by Sanger sequencing using the primers given in table 11.

To generate the pMSCV mSmo^wt mCherry (see Appendix) plasmid the W113Y mutation of the pMSCV mSmo^W113Y mCherry plasmid was reversed to the wildtype (wt) sequence using the primers CRD_mut_F2/CRD_mut_R2 and the QuikchangeII-XL site-directed mutagenesis kit in accordance to the manufacturer’s instructions. The following PCR settings were used:

- 38 -

1x 10 x reaction buffer 1‘ 95°C

10 ng pMSCV mSmo^W113Y mCherry 50‘‘ 95°C

18 cycles 125 ng CRD_mut_F2 (forward primer) 50‘‘ 60°C

125 ng CRD_mut_R2 (reverse primer) 9‘ 50‘‘ 68°C 1 µl dNTP mix (proprietary solution) 7‘ 68°C

3 µl Quiksolution 2‘ 37°C

2.5 U PfuUltra HF DNA polymerase

After the PCR reaction the amplification product was restricted by DpnI and transformed to E. coli XL-10 Gold bacteria, clones were picked as described above and small-scale plasmid preparations were performed. The site-directed mutagenesis was verified by Sanger sequencing using the primers mSmo_D54F and CRD_R. After medium-scale plasmid preparation of one positive clone, the integrity of the Smo sequence was verified by Sanger sequencing and the primers given in table 11.

The plasmid pMSCVpuro mSmo^ΔCRD mCherry (see Appendix) was generated according to the sequence described by Nedelcu et al. (Nedelcu et al. 2013). The CRD-deletion was achieved by performing a two-step overlap-extension PCR. First, two fragments were generated using the primer-combination 1A2F/1AR (206 bp) and 1BF/1BR (2,899 bp) using the following conditions in a 20 µl reaction volume for each PCR reaction:

overlap extension small fragment

50 ng pMSCV mSmo^W113Y mCherry Hotstart 98°C

0.5 µM SmoKlon 1A2F (forward primer) 30'' 98°C 0.5 µM SmoKlon 1AR (reverse primer) 8'' 98°C

35 cycles

0.2 mM dNTP mix 20'' 60°C

1x HF buffer 15'' 72°C

0.05 U Phusion High-fidelity polymerase 10' 72°C

∞ 8°C

overlap extension large fragment

50 ng pMSCV mSmo^W113Y mCherry Hotstart 98°C

0.5 µM SmoKlon 1BF (forward primer) 30'' 98°C

0.5 µM SmoKlon 1BR (reverse primer) 8'' 98°C

35 cycles

0.2 mM dNTP mix 20'' 65°C

1x HF buffer 50'' 72°C

0.05 U Phusion High-fidelity polymerase 10' 72°C

∞ 8°C

The amplification products were validated on a 1 % agarose gel and the overlap-extension-PCR was performed using the 1A2F/1AR (206 bp) and 1BF/1BR (2,899 bp) amplification products as template for the following protocol:

Methods

- 39 -

overlap extension PCR

2 µl small fragment PCR Hotstart 98°C

2 µl large fragment PCR 30'' 98°C

0.5 µM SmoKlon 1BF (forward primer) 8'' 98°C

35 cycles 0.5 µM SmoKlon 1BR (reverse primer) 20'' 65°C

0.2 mM dNTP mix 1' 72°C

1x HF buffer 10' 72°C

2.5 mM MgCl2 ∞ 8°C

0.05 U Phusion High-fidelity polymerase

The amplification products were separated on a 0.5 % agarose gel and the 3,077 bp fragment was excised and purified as described in chapter 5.1.3.3. Afterwards, the fragment were digested with BclI and NotI and cloned into pMSCVpuro vector linearized with BclI and NotI. The CRD deletion was confirmed by Sanger sequencing using the primer CRD_R. A deletion at position 1,636 was repaired using the QuikChangeII XL site-directed mutagenesis kit and the primers CRD_Ins_1636_F/CRD_Ins_1636_R.

5.2. Cell biology methods

5.2.1. Culture of eukaryotic cells

All eukaryotic cell lines were cultured in an incubator with constant 37 °C, 5 % CO2

and 95 % humidity. The media were refreshed every 3-4 days and the cells were splitted when reaching 80 to 90 % confluence. Splitting of the cells was conducted by detaching the cells with 1-3 ml of TrypLE express or for ASZ001 cells with 2-3 ml of trypsin/EDTA. After the cells started to detach, the reaction was stopped by the addition of FCS-containing medium and an appropriate volume was transferred to a new culture plate containing fresh medium.

The culture media and supplements are listed in table 7.

5.2.2. Cryopreservation of eukaryotic cells

For long-term storage of eukaryotic cell lines the cells were stored in liquid nitrogen.

For this purpose, 1-3 10 cm culture plates with 90 % cell confluency were rinsed with 1 x PBS and detached as described above. The detached cells were transferred into a 15 ml reaction tube und pelleted by centrifugation at 300 x g for 5’ and 4°C. Afterwards the cells were resuspended in 10 ml culture medium supplemented with 10 % DMSO and appropriate

- 40 -

antibiotics. 1 ml aliquots were frozen in Cryovials at -80°C in the freezing device Mr.

Frosty^TM for 16 h. Afterwards, the cells were stored in liquid nitrogen.

To thaw the cells they were rapidly warmed and transferred to 10 ml 1 x PBS, pelleted as described above and resuspended in fresh culture medium. Cells were transferred to a 5 or 10 cm culture dish and stored in an incubator. The next day the medium was replaced with fresh medium to ensure the complete removal of DMSO.

5.2.3. Transfection of eukaryotic cells

For the transfection of plasmid DNA, the cells were seeded as described in the respective experiments at a density of approximately 70 %. The cells were transfected using RotiFect^® according to the supplier’s instructions. For each cell line a specific ratio of DNA to RotiFect^® and a specific transfection time was used. In brief, one day after seeding, the transfection mixture was prepared by pipetting the appropriate volumes of DNA and RotiFect to culture medium without any supplements. This mixture was thoroughly vortexed to allow for forming complexes for 40’ at RT. Afterwards an appropriate volume of culture medium supplemented with 10 % FCS was added and mixed by gentle pipetting. This solution was added to cells, which were rinsed before with 1 x PBS. After the transfection time the solution was aspirated, the cells were rinsed with 1 x PBS and normal culture medium was added. If the transfection solution was not removed, the same volume of normal culture medium (including 1 % Penicillin/Streptomycin) was added to the cells after 6 h to prevent microbial contamination.

Table 16: Transfection conditions

cell line well type plasmids [µg]

Methods

- 41 -

5.2.4. Retroviral transduction of eukaryotic cells

For retroviral transduction of murine cells Platinum-E cells were used as packaging cell line. The latter cells were seeded at a confluency of 50-60 % in 5 cm culture dishes one day before transfection with the retroviral vectors. For the transfection the following reagents were mixed in the given order:

400 µl medium of the target cell line without supplements

5 µl RotiFect®

2.5 µg retroviral expression vector

The reagents were mixed by tapping and were incubated at RT for 30’. Hereafter, the Platinum-E cells were washed and the medium was replaced with 4 ml fresh medium used for the target cell line. The transfection mixture was added drop wise onto the plates and mixed by swirling. After 48 h the cell supernatant (Platinum-E conditioned medium; Platinum-E-CM) containing the viruses was sterile-filtered using a 0.45 µm pore sterile filter. 3 ml

The reagents were mixed by tapping and were incubated at RT for 30’. Hereafter, the Platinum-E cells were washed and the medium was replaced with 4 ml fresh medium used for the target cell line. The transfection mixture was added drop wise onto the plates and mixed by swirling. After 48 h the cell supernatant (Platinum-E conditioned medium; Platinum-E-CM) containing the viruses was sterile-filtered using a 0.45 µm pore sterile filter. 3 ml

Im Dokument Interaction of the Hedgehog and vitamin D receptor signaling pathways in Patched associated cancers (Seite 43-0)